The expression of foreign proteins by bacteria, yeast or mammalian cell lines has become routine. One type of commonly used means involves the construction of virion-plasmid hybrid vectors that possess the capacity to express cloned inserts in mammalian cells. The expression of the cloned gene with such hybrid vectors can occur in a transient, extrachromosomal manner, but higher production is usually obtained through random insertion of the vector into the host cell genome. The typical mammalian expression vector will contain regulatory elements, usually in the form of viral promoter or enhancer sequences and characterized by a broad host and tissue range, a polylinker sequence facilitating the insertion of a DNA fragment within the plasmid vector, and the sequences responsible for intron splicing and polyadenylation of mRNA transcripts. This contiguous region of promoter-polylinker-polyadenylation site is commonly referred to as the transcription unit. Viral promoter and enhancer regions have long been utilized as regulatory elements for use in mammalian host cells. For example, the strength of the CMV enhancer caused it to be a suggested component in eukaryotic expression vectors upon its discovery (Boshart et al., Cell, 41 (2):521-30 (1985)) and it has been utilized as a universal cell control element in transgenic mice (Schmidt et al. Mol. Cell. Biol. 10: 4406-4411 (1990)). The MPSV promoter coveys a wide host cell specificity to the virus including fibroblasts and hematopoietic stem cells (Stocking et al. Proc. Natl. Acad. Sci. USA, 82: 5746-5750 (1985)). Accordingly, this promoter has been used to express heterologous genes in a number of cell types, including skin fibroblasts (Pamer et al., Blood, 73: 438-445 (1989), primary hepatocytes (Ponder et al., Hum. Gene Ther. 2:41-52 (1991), and rodent cells lines and human fibroblast cell lines (van den Wollenberg, Gene 144: 237-241 (1994)).
Generally, there are two types of expression vectors suitable for use in eukaryotic cells, retrovirally-based systems and virion-plasmid hybrids described above. van den Wollenberg et al. describe a retroviral vector that comprises the CMV enhancer genetically engineered within the U3 region of the MPSV promoter. However, retroviral vectors have significant drawbacks for use in industrial level protein production. First, the level of protein production is severely hampered by the retroviral packaging sequence, a necessary component of such vectors, as it interferes with translational initiation. Second, protein production is reduced because the transport of retroviral messenger RNA is less efficient than a standard mRNA and there is competition between retroviral packaging and translation. Third, it is impossible to reach the gene copy numbers routinely achieved by standard vectors with an amplifying selection marker, due to the fact that a retroviral vector implants two promoters for each random integration, thus randomly activating downstream sequences with deleterious effects to the cell. Fourth, there are serious safety concerns with large-scale production of retroviral cultures due to random recombination to replication competency. Finally, retrovirally-established cell lines are harder to document and less efficient to develop since a viral production cell line must first be used to make a master cell bank, then the actual production cell line is produced, requiring a second round of analysis and banking. Accordingly, industrial production of protein is not routinely performed with retroviral vectors.
Thus, the expression of foreign proteins in commercially acceptable quantities remains a challenge. This is especially true in mammalian cell lines. Very often expression of a mammalian protein in a mammalian cell line is required in order to mimic the native form of the protein in all respects: structure, catalytic activity, immunological reactivity, and biological function. Often glycosylation or other post-translational modifications are the key to the production of the desired form of the protein, and bacteria or yeast systems are unable to accomplish these modifications. Thus, there remains a need for improved plasmids that promote the production of mammalian proteins in commercially viable quantities within mammalian host systems.